Phosphomimetic Mutation at Ser165 of α-Tubulin Promotes the Persistence of GTP Caps in Microtubules

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, 257-272). Ser165 lies near the interface between adjacent αβ-tubulin heterodimers and helix H8, which contains Glu254, the catalytic residue in α-tubulin that hydrolyzes the exchangeable GTP in β-tubulin (β:GTP) and triggers MT depolymerization. It was hypothesized that S165D, a phosphomimetic variant of α-tubulin, perturbs the alignment of α:Glu254 with respect to β:GTP, thereby impairing its hydrolysis. Molecular simulations were performed with cryoEM structures of MTs (PDB ID: 3J6E) in which phosphomimetic S165D or control S165N had been substituted. Unlike native and S165N structures, the distance between S165D and α:Glu254 increased by 0.6 Å, while the distance between α:Glu254 and β:GTP decreased by 0.4 Å. Rotation of β:GTP by 4 Å occurred in the S165D variant, whereas β:GTP in the S165N control was unchanged from the native structure. Additionally, the S165D variant exhibited an altered pattern of H-bonding to β:GTP, including the loss of three H-bonds. The significance of these findings to β:GTP hydrolysis was analyzed in MCF-10A human breast cells treated with an antibody that detects GTP-bound tubulin. Compared with controls, GTP-tubulin signals were at higher levels in cells that ectopically expressed S165D-α-tubulin (TUBA1C) or had been treated with PKC activator DAG-lactone to induce phosphorylation of Ser165 in native α-tubulin. These findings support a model whereby conformational changes induced by Ser165 phosphorylation alter the spatial relationship between β:GTP and α:Glu254, thereby slowing GTP hydrolysis and promoting GTP caps.

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